This project is based on the premise that aberrant activation of the PI3K/AKT pathway is a common feature of the dysregulated signaling network in human breast cancers and is responsible for key aspects of the transformed phenotype. We have employed pharmacologic inhibitors of specific elements of this pathway to interrogate its function and used the information obtained to develop novel clinical strategies. We have previously shown that the Hsp90 protein chaperone is required for the folding and stability of HER2 and AKT. In the first funding period, we showed that natural product inhibitors of Hsp90 induce HER2 degradation and inhibit AKT activation in breast tumor cells with HER2 amplification. This leads to loss of Dcyclin expression, G1 arrest, and differentiation of these tumors, in tissue culture and in vivo and sensitizes them to taxanes and to inhibition of angiogenesis. This work led to phase 1 and phase 2 trials of the Hsp90 inhibitor 17-AAG, which has now been shown to have significant antitumor activity in patients resistant to Herceptin (trastuzumab). These results and those of others suggest that Herceptin-resistant tumors remain dependent upon HER2 signaling. We hypothesize that HER2 activation of PI3K/AKT signaling is necessary for their growth. We now propose to use experimental models of Herceptin-resistant breast cancer to determine the mechanism of this phenomenon, the role played by PI3K/AKT signaling, and the potential therapeutic value of Hsp90, PI3K, and AKT inhibitors in this setting. PI3K/AKT signaling is activated by a variety of mechanisms in breast cancer: HER2 amplification, PI3K mutation, and decreased PTEN function are among the most prominent. We now plan to use selective pharmacologic inhibitors of PI3K, AKT, and mTor to study the biochemical and functional consequences of PI3K/AKT activation in each of these settings, to determine the feasibility of inhibiting this pathway in vivo and to use these data to develop mechanism-based combination therapies that exploit the effects of inhibiting these targets.